Catabolism of Glucose and Fatty Acids by VirulentTreponema pallidum
NEAL L. SCHILLER AND C. D. COX*
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01002
Received for publication 22 November 1976
We describe a procedure which permits essentially full recovery of physiologi-cally active Treponema pallidum from rabbit testicular extracts and greatlyreduces contaminating tissue material. Such preparations were employed forinvestigations of the ability of T. pallidum to catabolize glucose and fatty acids.Radiorespirometric studies revealed that glucose and pyruvate, but not oleate orpalmitate, could be degraded to CO2. The use of differentially labeled glucose, inconjunction with enzymatic analyses, indicated that glucose was catabolized bya combination of the Embden-Meyerhoff-Parnas and hexose monophosphatepathways. Pyruvate was degraded to CO2 from only the carboxyl position,suggesting the absence of a functioning Krebs cycle; this was substantiated byadditional enzyme analyses and radiorespirometric experiments. Oleate andpalmitate were incorporated but not catabolized by /3-oxidation. Glucose, al-though catabolized, was not incorporated. The potential significance of thesefindings is discussed.
Numerous attempts have been made to culti-vate virulent Treponema pallidum in pure cul-ture, but a review of the literature indicatesthat this goal has not been achieved (24, 31).Although it is considered an anaerobe, Cox andBarber (7) have shown that T. pallidum con-sumes 02 at a rate similar to that of the aerobicspirochete Leptospira. Such 02 uptake was cya-nide sensitive, indicating a functioning cyto-chrome oxidase. Inhibition of this 02 uptake byazide, chlorpromazine, and amytal further sug-gested a functioning electron transport systemfor the oxidation of reduced nicotinamide ade-nine dinucleotide (NADH) to 02. The couplingof this system to oxidative phosphorylation wassuggested. The possible aerobic nature of T.pallidum was recently supported by Basemanet al. (3), who found that glucose degradationand protein synthesis proceeded optimally in 02concentrations of 10 to 20% and were inhibitedunder anaerobic conditions.The purpose of this investigation was to con-
tinue to examine the physiology of this spiro-chete and, in particular, to determine the possi-ble catabolic activities of virulent T. pallidumfor glucose and fatty acids as potential oxidiza-ble substrates.
MATERIALS AND METHODSBacteria. The virulent Nichols strain of T. palli-
dum was used throughout this study. Cultivation ofthis organism in rabbits has been previously de-scribed (7). Testicles from exsanguinated, infectedrabbits were extracted either in a medium consist-
ing of 0.075 M sodium citrate containing 10% (vol/vol) inactivated pooled rabbit serum (Pel-Freez,Inc., Rodgers, Ark.) (7) or in an aqueous solution of0.01 M Na2HPO4, 0.14 M NaCl, and 0.06% reducedglutathione, adjusted to pH 7.3 with 0.1 N NaOH(PBS-G) (4). The extraction procedure was similar tothat previously described (11). Each testicle wastrimmed of fat and of the dorsal vein, cut lengthwise,and the edges of each half were snipped severaltimes with scissors. The testicular tissue was thenextracted in the appropriate medium (10 ml/testicle)in an atmosphere of 95% N2 and 5% CO2 by rotationat 70 rpm for 1 h at 25°C, followed by 1 h at 4°C.These extracts were adjusted for cell density in theextraction medium.
In previous experiments, some of which we havereported (7), treponemes extracted from tissue bydifferential centrifugation consumed 02 at levelssignificantly higher than animal-cell controls frominfected tissue. Treponeme and animal-cell prepara-tions were established by procedures previously out-lined by us (7) and later described by Nichols andBaseman (20). In the present studies tissue extractswere centrifuged at 300 x g for 10 min to remove themajority of contaminating tissue in the low-speedpellet (LSP). The supernatant fluid (LSS) was fil-tered through Nucleopore filters (see Results), andthe filtered suspension of treponemes was centri-fuged at 17,000 x g for 30 min. Treponemes in thehigh-speed pellet (HSP) were resuspended in a smallamount of high-speed supernatant fluid (HSS) andused in incorporation and radiorespirometric experi-ments. Enzyme assays were performed on sonicallytreated treponemes from HSP. The tissue-cell con-trols consisted of tissue cells from LSP resuspendedin HSS to a concentration of 104/ml, or about 500times higher than detectable levels in the filtered
treponemal suspension or the HSP. The other con-trol consisted of HSS to reflect soluble or membrane-associated enzyme activity resulting from the fewtissue cells and/or treponemes. Tissue-cell controlswere used to monitor and correct values in incorpo-ration studies, and both tissue-cell and HSS controlswere used similarly in metabolic experiments.
Incorporation experiments. PBS-G was selectedas the extraction medium since it was free fromglucose and fatty acids, thereby enhancing thechance of detecting any uptake of "4C-labeled sub-strate added to this medium.
After the addition of isotopically labeled com-pounds to treponeme suspensions, the samples wereswirled on a platform rotator at 120 rpm at 25°C.Preliminary experiments in several metabolic stud-ies indicated no substantial differences among thoseperformed at 25, 30, or 37°C, and room temperatureof 250C was chosen for these and subsequent experi-ments. At selected time intervals, samples of cellsuspensions were removed, incubated at 56°C for 5min, and centrifuged at 30,000 x g for 30 min. Thecells were washed in PBS-G and centrifuged as be-fore, and the pellet was suspended in absoluteethanol and transferred to scintillation vials. Afterevaporation, the residue was resuspended in scintil-lation fluid, and radioactivity was determined in aBeckman LS-100 liquid scintillation counter. Theefficiency of counting in these experiments was 95%.The scintillation fluid contained 700 ml of scintilla-tion-grade toluene, 300 ml of absolute ethanol, 0.6%2,5-diphenyloxazole (PPO), and 0.1% p-bis-(o-meth-ylstyryl)-benzene (bis-MSB).
In some experiments uptake of radioisotopes wasterminated by the addition of trichloroacetic acid.The cells were collected by centrifugation andwashed once with PBS-G. They were then washed incold 5% trichloroacetic acid, suspended, and trans-ferred to scintillation vials as described above.
Radiorespirometric experiments. The extractionfluid contained 75 mM sodium citrate and 10% (vol/vol) heat-inactivated pooled rabbit serum. Assayswere performed in a 10-ml Erlenmeyer flask sealedwith a rubber serum stopper, with a plastic centerwell (Kontes Glass Co.) suspended from the stopper.After 1.8 ml of the appropriate solution or suspen-sion was added, the flask was stoppered, 0.2 ml ofHyamine hydroxide was injected into the centerwell, and 0.2 ml of the isotope solution was added tothe solution or suspension. The flasks were incu-bated at 250C and rotated on a platform shaker at 70rpm. At the end of the reaction period of 2 h, 0.2 mlof 3 M H2SO4 was added to the reaction mixture torelease any soluble C02, and the flasks were incu-bated for an additional 60 min. The flasks were thenopened. The cup was immediately placed into 15 mlof scintillation fluid and radioactivity was mea-sured. Total glucose present was determined withthe Glucostat assay (Worthington BiochemicalsCorp.).Enzyme assays. Testicles were extracted in PBS-
G, and the second extraction was performed withfresh PBS-G for 1 h at 25°C. The HSP was washed in10 ml of 0.02 M sodium phosphate buffer (PBS), pH7.2, resuspended in a small volume of PBS, andeither frozen or used immediately.
Cells were disrupted by sonic treatment in a Ray-theon Sonifier using the minimal exposure neces-sary for the disappearance of all intact treponemesas observed by dark-field microscopy. At least three1-min bursts were usually required. Cellular debriswas removed by centrifugation for 20 min at 12,000x g, and the supernatant fraction was used as asource of soluble enzymes. The pellet was resus-pended in PBS and used as a source of membrane-associated enzymes. Extracts were kept at 4°C andused immediately for enzyme assays. Protein wasdetermined by the Folin phenol method (15). Assayswere made on extracts containing 10 to 100 ,ug ofprotein.
Most enzyme assays involved coupling the spe-cific reaction to be determined to a pyridine nucleo-tide-dependent system and, unless otherwise noted,followed the general procedures described in Meth-ods in Enzymology, vol. 1 (6). Oxidation or reductionof pyridine nucleotides was followed at 340 nm in aGilford model 240 recording spectrophotometer. Atotal volume of 0.15 ml was used in each cuvetteunless otherwise noted. One unit of enzyme activitywas defined as the amount of enzyme resulting inthe oxidation or reduction of 1 ,umol of pyridinenucleotide per min at 25°C. Specific activities areexpressed in terms of units of enzyme per milligramof crude treponemal extract protein. Control cu-vettes lacking various components of the specificassay, or treponemal extract, or including enzymesfrom other sources were included for each assay.Enzymes of the citric acid cycle were determined
in the following manner. The malate dehydrogenase(EC 1.1.1.37) assay reaction mixture contained 10.0,umol of tris(hydroxymethyl)aminomethane (Tris)buffer (pH 8.0), 2.0 ,umol of potassium oxalacetate,1.0 gmol of MgSO4, 0.075 ,imol of NADH, and 1.0,unmol of 2-mercaptoethanol. A reaction mixture formeasuring citrate synthase (EC 4.1.3.7) was em-ployed which required the presence of malate dehy-drogenase in the extract. This enzyme system com-bined 10.0 ,umol of Tris buffer (pH 8.0), 2.0 gtmol ofsodium malate, 0.3 ymol of acetyl coenzyme A (ace-tyl-CoA), 1.0 ,umol of MgSO4, 0.075 ,umol of nicotina-mide adenine dinucleotide (NAD), 1.0 ,umol of 2-mercaptoethanol, and pig heart malic dehydrogen-ase. Fumarate hydratase (EC 4.2.1.2) activity wasestimated by replacing sodium malate in the priorassay with potassium fumarate. Fumarate hydra-tase was also assayed according to a procedure de-scribed by Massey (17) which was based on the highultraviolet absorption of fumarate. This assay mix-ture contained 2.0 ,mol of potassium fumarate, 0.02M sodium phosphate buffer, pH 7.2, and cell-freeextract in a total volume of 0.15 ml. The consump-tion of fumarate was monitored by following thedecrease in absorbance at 300 nm. Isocitrate dehy-drogenase (EC 1.1.1.42) assay mixture contained10.0 ,imol of Tris buffer (pH 8.0), 2.0 ,umol of sodiumisocitrate, 1.0 ,umol of MgSO4, and 0.075 ,umol ofnicotinamide adenine dinucleotide phosphate(NADP). Sodium citrate substituted for isocitrate inassays for aconitate hydratase (EC 4.2.1.3). Oxoglu-tarate dehydrogenase (EC 1.2.4.2) reaction mixturecontained 10.0 ,mol of Tris buffer (pH 8.0), 2.0 ,umolof sodium oxoglutarate, 0.075 zmol of NAD, and 0.2
,umol of coenzyme A (CoA). Succinic dehydrogenase(EC 1.3.99.1) was assayed according to the proceduredescribed by Massey (18), with a 3.0-ml reactionmixture. Isocitrate lyase (EC 4.1.3.1) was measuredessentially by the method described by Reeves andAjl (23), with the following components in a 0.15-mlreaction mixture: 10.0 gmol of Tris buffer (pH 7.3),1.0 umoi of MgSO4, 1.0 gmol of phenylhydrazinehydrochloride, 0.1 ,umol of cysteine hydrochloride,and 2.0 ,umol of sodium isocitrate. The malate dehy-drogenase (decarboxylating) (NADP+) enzyme (EC1.1.1.40) was assayed essentially as described byOchoa (21) in a reaction mixture containing 10.0,umol of Tris buffer (pH 7.3), 1.0 ,mol of MgSO4,0.075 ,umol of NADP, and 2.0 ,tmol of either sodiummalate or potassium oxalacetate.
Glutamate dehydrogenase (EC 1.4.1.2) was as-sayed according to a procedure described by Strecker(25). When assayed in the forward direction, theassay mixture contained 10.0 umol of Tris buffer(pH 8.0), 0.075 ,umol of NAD, and 2.0 ,tmol of potas-sium glutamate. For the reverse reaction, the assaymixture contained 10.0 gmol of Tris buffer (pH 8.0),1.0 ,umol of NH4Cl, 0.075 ,tmol of NADH, and 2.0i,mol of sodium oxoglutarate.The acetyl-CoA acetyltransferase (EC 2.3.1.9) as-
say followed the general procedure described byHenneberry and Cox (12), with the following compo-nents in 0.15 ml: 10.0 ,umol of Tris buffer (pH 8.0),1.0 ,mol of MgSO4, 0.05 gmol of CoA, 0.075 ,mol ofNAD, 2.0 ,umol of sodium malate, 0.12 umol of ace-toacetyl-CoA, 1.0 ,umol of dithiothreitol, 0.92 unit ofpig heart citrate synthase, and 1.2 units of pig heartmalic dehydrogenase.Enoyl-CoA hydratase (EC 4.2.1.17) was measured
according to the procedure of Henneberry and Cox(12), with a reaction mixture containing 10.0 ,umolof Tris buffer (pH 8.0), 1.0 ,umol of dithiothreitol, 1.0,umol of MgCl2, 0.075 ,umol of NAD, 0.6 ,umol ofcrotonyl-CoA, and 0.04 unit of pig heart 3-hydroxy-acyl-CoA dehydrogenase (EC 1.1.1.35). The presenceof 3-hydroxyacyl-CoA dehydrogenase was deter-mined by employing the same assay mixture with-out the commercial enzyme.Enzymes of the glycolytic and pentose shunt path-
ways were also investigated. Glyceraldehyde-phos-phate dehydrogenase (EC 1.2.1.12) was measured ina reaction mixture containing 10.0 ,umol of Trisbuffer (pH 8.0), 0.075 ,umol of NAD, 2.5 ,umol ofsodium arsenate, 1.0 ,umol of 2-mercaptoethanol,and 2.0 ,umol of glyceraldehyde-3-phosphate. At-tempts were made to couple other enzymatic reac-tions to reduction of NAD via glyceraldehyde-phos-phate dehydrogenase. Fructose-biphosphate aldol-ase (EC 4.1.2.13) was assayed by replacing glyceral-dehyde-3-phosphate with fructose-1,6-diphosphate.Triosephosphate isomerase (EC 5.3.1.1) was assayedby replacing glyceraldehyde-3-phosphate with dihy-droxyacetone phosphate and by substituting trieth-anolamine buffer (pH 8.5) for Tris buffer. 6-Phos-phofructokinase (EC 2.7.1.11) was assayed withfructose-6-phosphate as the substrate, with the ad-dition of 0.75 ,umol of adenosine triphosphate (ATP)and 1.0 ,mol of MgSO4.
Glucose-6-phosphate dehydrogenase (EC 1.1.1.49)was assayed in a reaction mixture containing 10.0
,tmol of Tris buffer (pH 8.0), 1.0 ,umol of MgSO4,0.075 ,umol of NADP, and 2.0 /mol of glucose-6-phosphate. Phosphogluconate dehydrogenase (EC1.1.1.43) was estimated by replacing glucose-6-phos-phate with 6-phosphogluconate. The presence of glu-cose-6-phosphate dehydrogenase enabled the meas-urement of two enzymes by coupling these enzymesto the dehydrogenase assay. Hexokinase (EC2.7.1.1) was measured by replacing glucose-6-phos-phate with glucose and by adding 0.75 Amol of ATP.Glucosephosphate isomerase (EC 5.3.1.9) was as-sayed with 2.0 ,hmol of fructose-6-phosphate used assubstrate in place of glucose-6-phosphate.
Determinations of lactate dehydrogenase (EC1.1.1.27) involved a system containing 10.0 ,umol ofTris buffer (pH 8.0), 2.0 ,umol of sodium pyruvate,0.1 ,Lmol of ZnC12, 1.0 ,umol of MgSO4, and 0.075,umol of NADH. Pyruvate kinase (EC 2.7.1.40) wasdetermined by coupling pyruvate formation to lac-tate dehydrogenase-mediated consumption ofNADH. Reaction mixtures contained 10.0 ,umol oftriethanolamine buffer (pH 8.5), 2.0 umol of phos-phoenolpyruvate (PEP), 0.1 ,umol of ZnCl2, 1.0 ,umolof MgSO4, 0.75 ,umol of adenosine diphosphate(ADP), 1.0 ,tmol of 2-mercaptoethanol, and 0.075,umol of NADH. For determination of enolase (EC4.2.1.11) and phosphoglyceromutase (EC 2.7.5.3) ac-tivity, the pyruvate kinase assay mixture was sup-plemented with 2.0 umol of 3-phosphoglycerate and2.2 units of rabbit muscle pyruvate kinase, and PEPwas omitted.
In all assays where NADH consumption was mea-sured, activities were corrected for NADH dehydro-genase activity. NADH dehydrogenase (EC 1.6.99.3)was assayed in a reaction mixture c6ntaining 10.0,umol of Tris buffer (pH 8.0) and 0.075 ,umol ofNADH.The pyruvate dehydrogenase (EC 1.2.4.1) assay
was performed by a method described by Korkes (13)and contained 10.0 umol of Tris buffer (pH 8.0), 1.0,umol of MgSO4, 0.1 umol of CoA, 0.075 ,umol ofNAD, 1.0 ,umol of reduced glutathione, 0.1 ,umol ofthiamine pyrophosphate, and 2.0 ,umol of sodiumpyruvate.
Pyruvate oxidase (EC 1.2.3.3) was assayed as de-scribed by Hager and Lipmann (10), and acetyl phos-phate formation was measured by the hydroxamicmethod of Lipmann and Tuttle (14).
Pyruvate dehydrogenase (cytochrome) (EC1.2.2.2) was determined by the procedure describedby Williams and Hager (32). 14CO2 was trapped andmeasured as described earlier for the radiorespiro-metric experiments.
Chemicals. [U-14C]oleate and [U-14C]pyruvatewere purchased from ICN Radioisotope Division; allother radioactive compounds were obtained fromNew England Nuclear Corp. All chemicals werereagent grade or the equivalent. Sigma ChemicalCo. was the source of all commercial enzymes usedin this study.
RESULTSRemoval of tissue cells from suspensions of
T. pallidum. Although the previous extractionprocedure (7) seemed adequate for 02 uptake
studies, the use of this preparation for sub-strate oxidation and enzymatic analyses was
considered unacceptable. Therefore, attemptswere made to free these treponemal prepara-
tions from contaminating tissue cells. Severalinvestigators have examined various tech-niques to obtain such preparations. Thesemethods include density gradient centrifuga-tion (22), continuous-flow zonal centrifugationin cesium chloride gradients (26), discontinuousgradients of sodium and meglumine diatri-zoates (4), and membrane filtration procedures(5). All of these techniques damaged the trepo-nemes except the filtration procedure, whichremoved too many treponemes. However, J. W.Foster suggested the use of Nuclepore filters forthis purpose (personal communication), andseveral experiments were designed to investi-gate this possibility.
Nucleopore membranes ranging from 0.8- to5.0-,um pore sizes were examined in an effort toremove all observable tissue cells while allow-ing full recovery of the treponemes. Cells were
counted immediately before and after filtrationby use of a Petroff-Hausser counting chamberwith dark-field illumination. Either 25- or 47-mm diameter filters were routinely used withmild vacuum. The limit of cell detection, basedon the observation of 1 or 2 cells per entirecounting chamber, was between 5 x 103 and 1 x104 cells per ml. Both 0.8- and 1.0-,um Nucleo-pore filters consistently removed all observabletissue cells while allowing essentially full re-covery of T. pallidum (Table 1). The sensitivityof the counting procedure was increased by cen-trifuging the filtrate at 35,000 x g for 30 minand resuspending the pellet in smaller vol-umes. This procedure indicated that less than200 tissue cells per ml could have passedthrough the 0.8-,um filter. Larger pore sizes of3.0 and 5.0 ,um allowed spermatozoa, smalllymphocytes, and some red blood cells to passthrough the filters. However, prefiltrationthrough 3.0-jum filters permitted 50 to 80 ml oftreponemal suspension to pass through the 0.8-,um filter before plugging of the membrane oc-curred, compared with 30 to 50 ml without pre-
filtration. Filters of smaller pore sizes were nottested.The metabolic activity of the treponemes be-
fore and after filtration was examined by deter-mination of Q(02) values as described by Coxand Barber (7). As shown in Table 1, the meta-bolic activity ofthe treponemes was maintainedafter passage through 0.8- or 1.0-,um Nucleo-pore filters. Removal of tissue cells from trepo-neme suspensions without decreasing Q(02)values further supports the previous conclusion(7) that 02 consumption was associated with T.pallidum. Although the filtration procedurewas successful in removing essentially all tis-sue cells from the treponeme suspension, thepossibility remained that a few tissue cells andsoluble enzymes or membranes from tissuecells or treponemes might contribute to themetabolic activities observed. Thus, activitiesof the tissue-cell and HSS controls continued tobe routinely monitored.
Substrate incorporation studies. Several14C-labeled compounds were added to resting-cell suspensions of T. pallidum in an attempt todefine compounds which might serve as a majorsource of cell carbon. At indicated times, sam-ples were removed and the amount of incorpo-rated radioactivity was determined. [U-14C]glucose was not incorporated into eithermedium- or trichloroacetic acid-washed cellsduring a 2-h test period. Incorporation overlonger time periods was not examined becausethe motility of the treponemes decreased after 2h.Nelson (19) and Weber (30) have reported
that bovine serum albumin (BSA) prolongedtreponemal survival. It seemed reasonable thatthis might reflect a requirement for fatty acidspresent in BSA, and studies were performed todemonstrate the incorporation of fatty acids byT. pallidum. Since free fatty acids were precip-itated with cold trichloroacetic acid, determina-tions were performed only on medium-washedcells.
Incorporation of fatty acids into medium-washed cells was observed by using treponemesextracted in PBS-G and suspended in PBS-G
TABLE 1. Use ofNuclepore membranes to remove tissue cells from T. pallidum suspensionsFilter size Before filtration After filtration
(Am) T. pallidum Tissue cellsa Q(02)b T. pallidum Tissue cells Q(O2)0.8 1.5 x 108e 4.1 x 105 0.0293 1.5 x 108 <200 0.03461.0 2.1 x 108 1.8 x 106 0.0312 2.0 x 108 <5 x 103 0.03753.0 2.8 x 108 1.1 X 106 _ d 2.2 x 105 1 x105 -
a Tissue cells include red blood cells, white blood cells, and spermatozoa.b Q(02) values are expressed as microliters of 02 consumed per minute per 108 treponemes.e Values are expressed as cells per milliliter and represent an average of four determinations.d Not determined.
supplemented with [U-14C]oleate or [U-14C]palmitate (Table 2). Since the actualamount of unlabeled fatty acid contributed bytissue material during the extraction periodwas not known, it was not possible to expressthe results in terms of absolute amounts of fattyacids incorporated.
Substrate degradation. The ability of trepo-nemal suspensions to convert fatty acids toCO2, a process which would normally involvethe participation of the Krebs cycle, was exam-ined. Studies using [U-14C]palmitate, [U-'4C]oleate, or a mixture of these two isotopesfailed to demonstrate any radioactive CO2release, substantiating previous findings of Ni-chols and Baseman (20). These results sug-gested that T. pallidum may not degrade oleateor palmitate by a ,-oxidation pathway, or thatany two-carbon piece produced is not furtheroxidized by a functional Krebs cycle. The for-mer seems likely since enzymes of the ,-oxida-tion pathway, including acetyl-CoA acetyl-transferase, enoyl-CoA hydratase, and 3-hy-droxyacyl-CoA dehydrogenase, were not de-tected in the treponemal cell extracts.Although glucose was not incorporated into
T. pallidum cells, the possibility existed thatglucose was used as an energy source. Experi-ments with [U-14C]glucose confirmed the pre-vious observation of Nichols and Baseman (20)that [U-14C]glucose was degraded to 14CO2 by T.pallidum, and that the amount of 14CO2 re-leased was proportional to the number of trepo-nemes present.
Additional support for these findings was ob-tained by examining the rate of [U-14C]glucosedegradation by these treponemes (Fig. 1). '4CO2was released initially at a rapid rate and beganto plateau after 90 min. This plateau appearedshortly before treponemal motility, which was
TABLE 2. Uptake of [U-_4C]palmitate and [U-'C]oleate by T. pallidum
Amt (cpm) taken up/109 T pallidumIncorporation cellsatime (min)
[U-'4C]palmitate [U-'4C]oleate2 945b 7,605
60 3,261 13,741120 5,223 17,881
a Treponeme suspensions contained 5.67 x 109 T.pallidum cells/ml and either 1.08 ,Ci of [U-'4C]oleate/ml with a specific activity of 828 gCi/,umol or 0.94 ,uCi of [U-'4C]palmitate/ml with a spe-cific activity of 750 ,uCi/,umol. Absolute concentra-tions of fatty acids present in extraction fluid werenot known.
b These values have been corrected for any contri-bution of the tissue-cell control. Each value repre-sents an average of two determinations.
.09
c\j0U .oc-
uli( .0o
HSP
A LSP
* HSS3
--A MB60 120 1BC)
MINUTES
FIG. 1. Rate of '4CO2 release from [U-14C]glucoseby T. pallidum. The reaction mixture contained 0.73,umol of glucoselml. At zero time 0.105 ,uCi of [U-14C]glucose was added, and release of 14C02 wasassayed at specific time intervals. The HSP reactionmixture (1 ml) contained 109 T. pallidum cells sus-pended in HSS. Tissue-cell (LSP) and HSS controlswere prepared as described in the text. The HSPcontrol from noninfected rabbits gave activities simi-lar to these two controls. Each point represents anaverage of three determinations.
monitored throughout all the studies, began todecrease. The tissue-cell and HSS controls, asexpected, released minimal levels of 14CO2. Fur-thermore, these control values did not increasewith time, which inspires confidence in the con-clusion that metabolic activity is due to thetreponemes in the HSP, and that the harvest-ing and processing procedures using filtrationwere successful in removing contaminating tis-sue material.Metabolism of glucose. The mechanisms of
glucose degradation were investigated with dif-ferentially labeled glucose, based on the char-acteristic patterns of CO2 release expected fordifferent major metabolic pathways in radiospi-rometric experiments (29). Table 3 is a compos-ite of four representative radioactive tracerstudies, extending the previous observation ofNichols and Baseman (20). The results indi-cated that roughly equal amounts of '4CO2 werereleased from [3-14C]- and [4-14C]glucose, whichsuggests degradation of glucose by the Emb-den-Meyerhoff-Parnas (EMP) pathway. If glu-cose were completely degraded to CO2 througha combination of the EMP pathway and theKrebs cycle, CO2 should be released equallyfrom carbons one and six of glucose. Resultspresented in experiments A and B (Table 3)indicate that glucose was not completely oxi-dized by T. pallidum.The release of "4CO2 from [1-14C]glucose could
be explained either by dissimilation via theEntner-Doudoroffmetabolic route or the hexosemonophosphate (HMP) pathway. The formerwould be implicated if the CO2 released from [1-14C]- and [4-14C]glucose were equivalent. Al-though the data presented in experiment D
(Table 3) might suggest this possibility, experi-ment C and similar repeated experiments indi-cated that the Entner-Doudoroff pathway was
not utilized by virulent T. pallidum.The involvement of the HMP pathway was
suggested by additional experiments examin-ing the rate of 14CO2 release from several 14C_labeled substrates. 14CO2 release from [1-'4C]glucose was clearly greater than that from[3,4-14C]glucose or [6-'4C]glucose, and indicatedthe importance of the HMP pathway in glucosecatabolism. These data further suggested theabsence of a complete Krebs cycle since therewas only minimal 14CO2 release from [6-4C]glucose. The absolute or relative amounts of14C02 released from [1-14C]- and [3,4-14C]glucosewere different in several experiments, but con-
sistently suggested an involvement of both theHMP and EMP pathways for the catabolism ofglucose.Pyruvate metabolism. The observation of
minimal 14CO2 release from [2-14C]_ or [6-'4C]glucose strongly suggested an incompleteoxidation of glucose. Several experiments weredesigned to examine the metabolism of pyru-vate, a probable intermediate of both the HMPand EMP pathways, and to determine whetherT. pallidum possessed a functioning Krebs cy-
cle. C02 was preferentially released from thecarboxyl position of pyruvate, as previously re-
ported by Nichols and Baseman (20). However,the inability ofT. pallidum to release CO2 fromC2 or C3 of pyruvate again suggested the ab-
sence of a complete Krebs cycle. Furthermore,T. pallidum suspensions or cell-free extractsdid not evolve radiolabeled CO2 from either a-
[U-14C]ketoglutarate or [6-14C]citrate.Enzyme assays. Cell-free extracts were pre-
pared from treponeme suspensions which hadbeen extracted in PBS-G, a serum-free me-
dium, to eliminate the possibility of measuring
TABLE 3. Release of 14CO, from differentially labeled["4C]glucose by T. palliduma
a Reaction mixtures contained 0.672 pmol of glucose/ml,and the specific activity of the isotopes added was approxi-mately 0.360 ACi/pmol of glucose. Each value has beencorrected for counts contributed by the HSS and tissue-cellcontrols (which did not exceed 0.0006) and represents anaverage of four determinations.
b These values are determined by subtracting-counts of3-'4C from those of 3,4-'4C. Label in the latter was equallydistributed according to the commercial source (New Eng-land Nuclear Corp.).
TABLE 4. Specific enzymatic activities of T.pallidum cell-free extracts
NADH dehydrogenase(EC 1.6.99.3) .......... 0.144 0.046a Expressed as micromoles of pyridine nucleotide
formed per minute per milligram ofprotein. Individ-ual assay mixtures contained from 10 to 100 ,ug ofextract protein.
b No activity was detected. Specific enzyme activ-ities of 0.0002 could be measured by the techniqueemployed.
enzymes present in rabbit serum. A wash oftheHSP was included to minimize the amount ofmembrane material or tissue enzymes whichmight be present in the HSP. Preliminary ex-periments were performed on treponemal sus-pensions which had been washed two or threetimes. However, the second and third washesdid not remove much protein, and, since theloss of treponemal protein was to be avoided,one wash was routinely used for all enzymeassays reported. Treponemes appeared to beintact after one wash as determined by dark-field microscopy.Table 4 lists the activities of treponemal cell-
free preparations for enzymes involved in theEMP and HMP pathways. Activities were ob-served for all enzymes related to these two met-abolic routes which were tested. Most of theactivity observed appeared in the soluble en-zyme fraction with the exception of pyruvatekinase, which was primarily associated with
the membrane fraction. These results sup-ported the radiorespirometric data suggestingthat glucose could be catabolized by T. palli-dum by the EMP and HMP pathways.
Considerable interest was centered on thepossible absence of a Krebs cycle, and the en-zymes associated with this cycle were investi-gated. As shown in Table 5, only isocitratedehydrogenase and malate dehydrogenase ac-tivities were detected. The absence of isocitratelyase activity suggested that a glyoxalate shuntwas not present.
Extractions of treponemes from testes werecarried out under an atmosphere of 95% N2 and5% C02. Since activities of some Krebs cycleenzymes have been reported to be inducible byoxygen (2, 9), all enzyme assays were also con-ducted on preparations from treponemes ex-tracted under atmospheric conditions. The re-sults were essentially the same regardless of
TABLE 5. Krebs cycle and associated enzymeactivities of T. pallidum cell-free extracts
a Expressed as micromoles of pyridine nucleotideformed per minute per milligram ofprotein. Individ-ual assay mixtures contained from 10 to 100 ug ofextract protein.
b Coupled assay.c No activity was detected. Specific enzyme activ-
ities of 0.0002 could be measured by the techniqueemployed.
INFECT. IMMUN.
the extraction conditions, and Table 5 presentsdata obtained with treponemal preparations de-rived from aerobically processed testes.The possibility existed that low-molecular-
weight components in these crude extractsmight inhibit the activity of some Krebs cycleenzymes. Therefore, extracts were dialyzedovernight in 1,000 volumes of distilled water at40C, but these preparations did not acquire ad-ditional activity. In addition, treponemal ex-tracts did not inhibit the enzymatic activity ofextracts prepared from well-characterized mi-croorganisms, such as Leptospira biflexa strainB16 and Escherichia coli. The possibility re-mained that sonic treatment destroyed activityin these extracts, and several preparationswere made by freeze-thaw disruption. How-ever, this procedure failed to reveal any addi-tional activity. These results tend to confirmour earlier observations suggesting the absenceof a Krebs cycle. However, negative enzymeassays must be interpreted cautiously. Sensi-tivities of the assays to detect very low activi-ties could not be increased because of limitingprotein concentrations of not greater than 4 mgof crude treponemal protein/ml.
DISCUSSIONOne of the major problems confronting inves-
tigators working on virulent T. pallidum hasbeen to obtain preparations of treponemes freefrom contaminating tissue material. The pres-ent investigation utilized Nucleopore mem-branes in addition to differential centrifugationto free treponemal suspensions from contami-nating rabbit tissue cells below detectable con-centrations, while allowing essentially fully re-covery of physiologically active treponemes.Results with the two controls demonstrated theeffectiveness of this procedure and provided es-sential monitoring during experiments. Wesuggest that this procedure of differential cen-trifugation and filtration should be useful inobtaining very clean treponemal suspensions,without loss of treponemes or their physiologi-cal activity.
Substrate incorporation studies indicatedthat T. pallidum did not incorporate glucoseinto macromolecular or structural material.The 33-h in vivo generation time for T. palli-dum reported by Cumberland and Turner (8)might imply that a 2-h exposure was too brieftobe significant. Similar results have been re-ported by Nichols and Baseman (20), who ob-served little radioactive incorporation into tri-chloroacetic acid-precipitable material after in-cubation for 2 or 4 h with [U-14C]pyruvate orselectively labeled pyruvate. The possibility re-
mains that glucose might be incorporated intomacromolecular or structural material, butthat longer exposure times would be requiredfor detection.Although T. pallidum did not degrade fatty
acids through the 3-oxidative pathway, the evi-dence suggested that T. pallidum can incorpo-rate both [U-14C]oleate and [U-14C]palmitate.Vaczi et al. (28) have demonstrated that trepo-nemes contain a considerable amount of oleicand palmitic acids (14 to 20% of the cell dryweight). The ability of T. pallidum to incorpo-rate these fatty acids might obviate the require-ment of synthesizing them de novo. Further-more, incorporated fatty acids might provide asource of carbon for the synthesis of other fattyacids or structural materials.
Radiorespirometric studies using differen-tially labeled glucose indicated that 14CO2 wasreleased primarily from [1-14C]glucose and [3,4-14C]glucose. Further investigation demon-strated that "4CO2 was evolved equally from[3-14C]- and [4-'4C]glucose, which implied theparticipation of the EMP pathway. The largeramount of 14CO2 released from [1-_4C]glucosewas probably due to the involvement of theHMP pathway. The existence of both pathwayswas supported by the observation of enzymaticactivity for the major enzymes of these meta-bolic routes. The existence ofthe HMP pathwaywould provide a source of NADPH, which isrequired for fatty acid biosynthesis (16). In ad-dition, the HMP pathway can also furnish in-termediates, such as pentose and triose phos-phates, which may be required for biosynthesis(16). The EMP pathway can serve as a majorenergy-yielding pathway for T. pallidum. Thepresence of excess pentose or triose phosphatesmay serve to regulate the demand for the HMPpathway, whereas energy requirements maycontrol the flow of the EMP pathway. The ob-servation of different activity levels ofEMP andHMP pathways during this investigation mayreflect this activity. Identification of controlmechanisms will most likely await growth ofthe treponemes in pure culture.The tracer studies suggested the absence of a
complete Krebs cycle. This conclusion was sup-ported by the following observations: (i) an in-complete oxidation of glucose, (ii) the preferen-tial degradation of [1-_4C]pyruvate with essen-tially no degradation of [2-_4C]- or [3-14Clpyruvate, (iii) the inability of T. pallidumto evolve 14CO2 from radiolabeled cycle interme-diates, and (iv) the absence of enzymatic activi-ties for most Krebs cycle enzymes. The absenceof malate dehydrogenase (decarboxylating)(NADP+) and fumarate hydratase would sug-
gest the absence of an incomplete Krebs cycleas seen in E. coli (2) or Listeria monocytogenes(27). Malate dehydrogenase (decarboxylating)(NADP+) is a reversible enzyme which canform oxalacetate or malate from pyruvate andcould theoretically serve as an entry into theKrebs cycle. However, no activity with thisenzyme was detected with either oxalacetate ormalate as substrate.The ability of the treponemes to degrade py-
ruvate to CO2 has already been described; how-ever, the mechanism of this degradation is notknown. Pyruvate dehydrogenase (EC 1.2.4.1), amultienzyme complex which requires lipoicacid for activity, was not detected. Pyruvateoxidase (EC 1.2.3.3) was measured according tothe procedure of Hager and Lipmann (10), whohad detected this enzyme in Lactobacillus del-brueckii. The inability to detect pyruvate oxi-dase is consistent with the study conducted byAjello (1), who demonstrated the inability ofvirulent T. pallidum to form active acetate.Ajello could not detect acetate kinase (EC2.7.2.1) or phosphate acetyltransferase (EC2.3.1.8) in her preparations. These resultswould suggest that T. pallidum cannot deriveATP from the conversion of pyruvate to ace-tate. The possibility that T. pallidum catalyzedthe oxidative decarboxylation of pyruvate toacetate and CO2 by use of a flavoprotein-cyto-chrome system was investigated. Such a sys-tem has been described by Williams and Hager(32) for E. coli. However, extracts of T. palli-dum were unable to demonstrate pyruvate de-hydrogenase (cytochrome) (EC 1.2.2.2) activityby this assay procedure. The mechanism of py-ruvate degradation by T. pallidum remainsunclear.From the results presented here, T. pallidum
would seem to have limited energy resources.Only two equivalents ofATP would be expectedfrom substrate-level phosphorylation of stepsthat occur during glucose degradation by theEMP pathway. The results of Cox and Barber(7) suggest that T. pallidum has an electrontransport system coupled to 02, and hence mayderive additional energy from oxidative phos-phorylation. Further investigation of a termi-nal electron transport system in T. pallidum isrequired. However, without a functioningKrebs cycle, our knowledge of their energy-yielding capability would still be consistentwith the slow generation time which has beenobserved for T. pallidum.
ACKNOWLEDGMENTS
This investigation was supported by Public Health Ser-vice grant AI-10982 from the National Institute of Allergy
and Infectious Diseases. The senior author was a predoc-toral trainee on grant GM-02168 from the National Instituteof General Medical Sciences.
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